In the proposed research cytochrome c (cyt c) will be studied as a model self antigen to determine how intracellular antigens become available for B lymphocyte recognition in tolerance and autoimmunity. and, in mice, there appears to be tolerance to a minor antigenic site on cyt c but not to the major site of native mouse cyt c. During necrosis, native cyt c released from the cells could serve as an immunogen. Cyt c has been shown to translocate from mitochondria during apoptosis and may eventually be exposed at the cell surface or released from dying cells. Preliminary evidence does indicate the appearance of cyt c in blebs of apoptotic cells, a common fate for intracellular autoantigens. Although cyt c does not appear to be accessible at the cell surface in the blebs, an altered form of cyt c which may be relevant for B cell recognition has been isolated from the culture fluid of an apoptotic transformed cell line. Additional studies of a variety of cell types will be carried out to identify similarities and differences among them in the fate of cyt c in cell death. Since in vivo apoptotic cells and debris are engulfed by phagocytic cells, the effect of peritoneal macrophages on the appearance of extracellular cyt c will be examined in vitro. Whether an altered form of cyt arising in either apoptotic or necrotic cell death impacts on the B cell recognition of cyt as a tolerogen or autoantigen will also be studied. Anti-cyt c Ig-transgenic mice will be prepared and their fate in response to native cyt c (tolerance versus anergy) and to apoptotic/necrotic forms will be examined in vitro as will their fate in vivo. The reactivity of the various forms of cyt c with autoantibodies that may arise in autoimmune-prone mice will be analyzed to determine whether the autoantibodies may have bene elicited against native cyt c or a "death form." Finally, monoclonal antibodies reactive with any "death form" of cyt c will be prepared for use as reagents to identify altered cyt c in situ. The findings of this study will likely have implications for understanding the fate of other intracellular antigens and how it impacts in B cell recognition of the endogenous antigens.